Mycoplasmas are parasites of the respiratory epithelium and urogenital tract. Although mycoplasma infections are typically asymptomatic in mammals, they seem to be co-factors in diseases, such as AIDS (Acquired Immunodeficiency Syndrome), and in sequelae after mycoplasma infections having an autoimmune basis.
Mycoplasmas are the smallest self-replicating microorganisms and have unique properties among the prokaryotes, such as (i) their need for cholesterol to maintain their membrane envelope and (ii) the absence of an external wall. Mycoplasmas are known to cause pulmonary infection in humans. See, Razin et al., “Molecular biology and pathogenicity of mycoplasmas,” Microbiol. Mol. Biol. Rev.; 62(4):1094–1156, (1998). Furthermore, it is widely known that mycoplasmas can cause disease in most animals, including animals of commercial importance to the husbandry industry, such as cattle, swine, and fowl. See, Maniloff et al. Eds., Mycoplasmas, Molecular Biology and Pathogenesis, American Society for Microbiology (Washington, 1992).
It has been suggested that mycoplasma may play a role in the pathogenesis of a number of human diseases, including asthma, diseases of the large intestine, rheumatoid diseases such as rheumatoid arthritis, maculopapular erythemas, stomatitis, conjunctivitis, pericarditis, Alzheimer's Disease, multiple sclerosis, the sequelae of AIDS and HIV infection, genito-urinary infections, diseases of chronic fatigue like Chronic Fatigue Syndrome, and Gulf War Syndrome. However, the actual role of mycoplasmas in these various diseases have been difficult to determine, because most of the associations drawn to mycoplasma infection are based on serologic evidence rather than direct observation of mycoplasma organisms in disease lesions. See, Cole, “Mycoplasma interactions with the immune system: implications for disease pathology,” (http://www.compkarori.com/arthritis/pi16002.htm); Cole, “Mycoplasma-induced arthritis in animals: relevance to understanding the etiologies of the human rheumatic diseases,” Rev. Rhum. Engl. Ed.; 66(1 Suppl):45S–49S (1999); and Nicolson et al., “Mycoplasmal infections in chronic illnesses,” (http://www.gulfwarvets.com/article24.htm).
Mycoplasma as well as chlamydia have been implicated in vascular disease, but the etiologic relationships have not been confirmed. See, Chen et al., “Carditis associated with Mycoplasma pneumoniae infection,” Am. J. Dis. Child. 140:471–472 (1986); Clyde et al., “Tropism for Mycoplasma gallisepticum for arterial walls,” Proc. Natl. Acad. Sci. U.S.A. 70: 1545–1549 (1973); Danesch et al., “Chronic infections and coronary artery disease: is there a link?”, Lancet 350:430–436 (1997); Farraj et al., “Mycoplasma-associated pericarditis, case report,” Mayo Clin. Proc. 72:33–36 (1997); Fu et al., “Middle cerebral artery occlusion after recent Mycoplasma pneumoniae infection,” J. Neurol. Sci. 157:113–115 (1998); Gurfinkel et al., “IgG antibodies to chlamydial and mycoplasma infection plus C-reactive protein related to poor outcome in unstable angina,” Arch. Inst. Cardiol. Mex. 67:462–468 (1997); Ong et al., “Detection and widespread distribution of Chlamydia pneumoniae in the vascular system and its possible implications,” J. Clin. Pathol. 49:102–106 (1996); Perez et al., “Leukocytoclastic vasculitis and polyarthritis associated with Mycoplasma pneumoniae infection,” Clin. Infect. Dis. 25:154–155 (1997); Taylor-Robinson and Thomas, “Chlamydia pneumoniae in arteries: the facts, their interpretation, and future studies,” J. Clin. Pathol. 51:793–797 (1998). In Maraha et al., “Is Mycoplasma pneumoniae associated with vascular disease,” J. Clin. Microbiol. 38:935–936 (February 2000), it was stated that “in a serological study, in contrast to C. pneumoniae antibodies, M. pneumoniae antibodies are not associated with recurrent events in patients with unstable angina”, citing Gurfinkel et al., supra. Maraha et al. reported that using PCR, they “were unable to detect M. pneumoniae in the great majority of the 103 tested specimens” of atherectomies and degenerative heart valves, and concluded that “the results . . . do not support the hypothesis that M. pneumoniae is an important factor in the development of vascular disease.” In contrast, Horne et al. have published a correlation between a positive serology for Mycoplasma pneumoniae and atherosclerosis (Horne et al., “IgA sero-positivity to Mycoplasma pneumoniae predicts the diagnosis of coronary artery disease,” J. Am. Coll. Cardiol. 35:321 (abstract) (2000)).
The co-occurrence of mycoplasma and other infectious agents seems to increase the virulence of both pathogens. For example, HIV patients, who have positive serology for Mycoplasma penetrans, are in worse clinical health than HIV patients who test negative for Mycoplasma penetrans. See, Blanchard et al., “AIDS-associated mycoplasmas,” Annu. Rev. Microbiol., 48:687–712, (1994).
Morphological studies of pathogenic mycoplasma indicate that these microorganisms which, unlike bacteria, lack cell walls, are strongly attached to the external surface of host cells through their membranes. This attachment is apparently the first step for colonization of a target tissue and a prerequisite for infection, as disclosed in Collier and Clyde, “Relationships between M. pneumoniae and human respiratory epithelium,” Infect. Immun., 3:694–701 (1971), and Kahane et al., “Attachment of mycoplasmas to erythrocytes: a model to study mycoplasma attachment to the epithelium of the host respiratory tract,” Isr. J. Med. Sci., 17:589–592 (1981). Moreover, experimental studies have demonstrated that mycoplasmas that were attached to macrophages could not be reached by different concentrations of complement, suggesting that cellular attachment may protect the mycoplasma, from the natural defense mechanisms of the host. See, Bredt et al., “Adherence of mycoplasmas: phenomena and possible role in the pathogenesis of disease,” Infection, 10(3):199–201 (1982), and Kahane, “Purification of attachment moiety: a review,” Yale J. Biol. Med., 53:665–669 (1983).
Accordingly, prevention of or interference with the first step of mycoplasma attachment can provide an important means of controlling infection. Currently existing antibiotics, however, have been ineffective at either preventing or breaking the adhesion of pathogenic mycoplasmas to the host cells.
The attachment zone of Mycoplasma pneumoniae (“M. pneumoniae ”) and of other mycoplasmas is rich in glycoproteins that contain sialic acid. See, Chandler et al., “Mycoplasma pneumoniae attachment: competitive inhibition by mycoplasmal binding component and by sialic acid-containing glycoconjugates,” Infect. Immun., 38(2):598–603 (1982), Glasgow and Hill, “Interactions of Mycoplasma gallisepticum with sialyl glycoproteins,” Infect. Immun.; 30:353–361 (1980), and Hansen et al., “Characterization of hemadsorption-negative mutants of Mycoplasma pneumoniae,” Infect. Immun., 32:127–136 (1981). Electron microscopy observations have indicated that glycoproteins linked to sialic acid mediate the attachment and the virulence of Mycoplasma pulmonis (“M. pulmonis”) in rats. See, Taylor-Robinson et al., “Mycoplasmal adherence with particular reference to the pathogenicity of Mycoplasma pulmonis,” Isr. J. Med. Sci., 17:599–603 (1981). Although mycoplasmas may attach to regions without the host cell sialic acid, the presence of sialic acid at the adhesion site may be essential for mycoplasmas to become virulent. See, Krause et al., “Identification of Mycoplasma pneumoniae proteins associated with hemadsorption and virulence,” Infect. Immun., 35:809–817 (1982), and Baseman et al., “Sialic acid residues mediate Mycoplasma pneumoniae attachment to human and sheep erythrocytes,” Infect. Immun., 38(1):389–391 (1982). This attachment zone is sensitive to pronase and can be inactivated by neuraminidase, as disclosed in Gabridge and Taylor-Robinson, “Interaction of Mycoplasma pneumoniae with human lung fibroblasts: role of receptor sites,” Infect. Immun., 25:455–459 (1979).
Sialic acid was initially discovered on the surface of Trypanosoma cruzi (“T. cruzi”) by Pereira et al. in 1980. See, Pereira et al., “Lectin receptors as markers for Trypanosoma cruzi. Development stages and a study of the interaction of wheat germ agglutinin with sialic acid residues on epimastigotes cells,” J. Exp. Med., 152:1375–92 (1980). Pereira also first demonstrated in 1983 that T. cruzi has sialidase activity. See, Pereira, “A developmentally regulated neuraminidase activity in Trypanosoma cruzi,” Science, 219:1444–46 (1983).
Trans-sialidase, an enzyme expressed on the T. cruzi's surface, catalyzes the transfer of sialic acid from host glycoconjugates to glycoprotein molecules on the surface of the parasite. See, Schenkman et al., “Attachment of Trypanosoma cruzi trypomastigotes to receptors at restricted cell surface domains,” Exp. Parasitol., 72:76–86 (1991). The enzyme is present both in the epimastigote form (i.e., in the invertebrate vector) and in the trypomastigote form (i.e., infectious form that circulates in the blood of the vertebrate host). See, Agusti et al., “The trans-sialidase of Trypanosoma cruzi is anchored by two different lipids,” Glycobiology, 7(6):731–5, (1997).
The catalytic portion of trans-sialidase (“TSC”) has two kinds of enzymatic activity: (1) neuraminidase activity, which releases sialic acid from the complex carbohydrates; and (2) sialil-transferase activity, which catalyzes the transfer of sialic acid from glyconjugate donors to terminal β-D galactose containing acceptors. See, Scudder et al., “Enzymatic characterization of beta-D-galactoside alpha 2,3-trans-sialidase from Trypanosoma cruzi” J. Biol. Chem., 268(13):9886–91 (1993).
In the complete native form of trans-sialidase (“TSN”), the enzyme has a C-terminal extension having a repetitive sequence of 12 amino acids previously identified as SAPA (i.e., Shed-Acute-Phase-Antigens). Although the repetitive sequence of amino acids is not directly involved in the catalytic activity, it stabilizes the trans-sialidase activity in the blood to increase the half-life of the enzyme from about 7 to about 35 hours. See, Pollevick et al., “The complete sequence of SAPA, a shed acute-phase antigen of Trypanosoma cruzi,” Mol. Biochem. Parasitol. 47:247–250 (1991) and Buscaglia et al., “The repetitive domain of Trypanosoma cruzi trans-sialidase enhances the immune response against the catalytic domain,” J. Infect. Dis., 177(2):431–6 (1998).
In the plasma membrane of T. cruzi trypomastigotes, the sialic acid acceptors are involved in the adherence of the parasite to the host and its subsequent invasion into the cell. Trans-sialidase may also sialylate the host cell glycoconjugates, forming receptors that will be used by the trypomastigotes for the attachment and penetration into the target cells.
The trans-sialidase enzyme of T. cruzi has been well characterized. See, Pollevick et al., Mol. Biochem. Parasitol. 47:247–250 (1991); Pereira et al., J. Exp. Med. 174:179–192; Schenkman et al., “Trypanosoma cruzi trans-sialidase and neuraminidase activities can be mediated by the same enzyme,” J. Exp. Med. 175:567–575 (1992); Schenkman et al., “Structural and functional properties of Trypanosoma trans-sialidase,” Annu. Rev. Microbiol. 48:499–523 (1994); Schenkman et al., “A proteolytic fragment of Trypanosoma cruzi trans-sialidase lacking the carboxy-terminal domain is active, monomeric, and generates antibodies that inhibit enzymatic activity” J. Biol. Chem. 269:7970–7975 (1994); Campetella et al., “A recombinant Trypanosoma cruzi trans-sialidase lacking the amino acid repeats retains the enzymatic activity,” Mol. Biochem. Parasitol. 64:337–340 (1994); Parodi et al., “Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi” EMBO J. 11:1705–1710 (1992); Uemura et al., “Only some members of a gene family in Trypanosoma cruzi encode proteins that express both trans-sialidase and neuraminidase activities,” EMBO J. 11:3837–3844 (1992); Cremona et al., “A single tyrosine differentiates active and inactive Trypanosoma cruzi trans-sialidase,” Gene 160:123–128 (1995).
The enzymatically active protein extracted from the T. cruzi trypomastigotes has 4 distinct amino acid regions: (1) a N-terminal region with approximately 380 amino acids of which 30% of the sequence is similar to bacterial sialidases; (2) a region with approximately 150 residues that does not show any similarity with any known sequence; (3) a region with homology to type III fibronectin (FnIII); and (4) a C-terminal region containing 12 repeated amino acids, which is the immuno-dominant portion and which is required for enzyme oligomerization. The N-terminal and the FnIII regions are important for trans-sialidase activity.
Native and purified trans-sialidase (“TS”) form multi-numerical aggregates having a molecular weight of more than 400 kDA. These aggregates are linked to the surface of the parasite membrane through a GPI anchor and are only released to the external medium through phospholipase C. After being denatured, the multi-numerical aggregates of TS migrate in a SDS-PAGE gel forming multiple bands ranging from about 100 to about 220 kDA.